FEMS Microbiology Letters 56 (1988) 135-138
Published by Elsevier
135
FEM 03354
The interaction of metronidazole with electron carriers
of the Azotobacter vinelandii respiratory particle fraction
Jay B. Peterson
Department of Botany, Iowa State University, Ames, IA, U.S.A.
Received 29 June 1988
Accepted 25 July 1988
Key words: Azotobacter vinelandii; Metronidazole; Flavin; Cytochrome
1. SUMMARY
Metronidazole, menadione bisulfite, and oxygen
oxidized NADH- and dithionite-reduced flavin of
the Azotobacter vinelandii respiratory particle fraction. The oxidation of NADH-reduced flavin by
metronidazole was slow compared to the oxidations by menadione bisulfite and oxygen.
Metronidazole caused spectral changes characteristic of cytochrome d oxidation, but the changes
in NADH-reduced particles were slow.
2. INTRODUCTION
Metronidazole (2-methyl-5-nitroimidazole-1ethanol) is an artificial one-electron acceptor of
low reduction potential ( - 4 8 5 mV at pH 7.0 [1]).
It has been used experimentally as a selective
inhibitor of low-potential processes. The compound inhibits nitrogenase activity in the aerobe
Azotobacter oinelandii with little effect on oxygen
uptake and no detectable effect on cell ATP, ADP
and AMP levels [2]. However, it slowly oxidizes
Correspondence to: Jay B. Peterson, Department of Botany,
Iowa State University, 353 Bessey Hall, Ames, IA 50011,
U.S.A.
whole-cell flavin to about the same extent as does
oxygen [3], suggesting that it is capable of oxidizing respiratory flavin. Because metronidazole is a
pharmaceutical used for treatment of infections by
anaerobic bacteria [4-7], there is great interest in
how it interacts with metabolism in aerobic and
anaerobic organisms.
This research was initiated to investigate the
specificity of metronidazole by examining its
oxidation of flavin and cytochrome electron carders of the respiratory particle fraction. Comparative studies were done with the electron acceptor
menadione bisulfite (MBS). MBS, unlike metronidazole, significantly inhibits oxygen uptake catalysed by respiratory particles [2]. The parent compound menadione is an electron acceptor of respiratory NADH dehydrogenase, a flavoprotein
[8].
3. MATERIALS AND METHODS
3.1. Bacterial growth and particle preparation
Azotobacter vinelandii strain OP (ATCC 13705)
was cultured and the R3 fraction containing respiratory membranes prepared as described [2].
Respiratory membranes were further purified by
passage through a 2.5 × 57 cm column of Sep-
0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies
136
harose CL-6B. The column buffer was 50 mM
potassium phosphate, p H 7.5 and the temperature
was 4 ° C. Fractions were 5 ml and three fractions
which contained the peak of N A D H oxidase and
malate-dependent oxygen uptake activities and a
peak of ravin fluorescence were pooled. These
were the void fractions (totally excluded). The
chromatography eliminated a peak of ravin of
lower molecular weight (less than 669000) and
increased the specific activities of the enzymes 4.4and 4.5-fold, respectively. N A D H oxidase specific
activity was 11.7 t~mol N A D H oxidized, rain -~
mg p r o t e i n - l at 23 ° C. Particle protein was measured by the method of Lowry et al. [9] after
trichloroacetic acid precipitation.
0o-
~
O-
Absorption-difference spectroscopy and fluorescence spectroscopy were as described [10].
Buffers used were 50 mM potassium phosphate,
p H 7.5.
3.3. Reagents
Reagents were purchased and used as described
[2,10]. Sepharose CL-6B and molecular-weight
standards were from Sigma Chem. Co., St. Louis,
MO. Metronidazole stock solutions were made up
as 0.5 M in DMSO.
630
~
~
C
4,~ ~
~I~0n~
500
0
O3
/ ' ~ [ ° 560 & AI 0,02
554~
_~_
m
.~
6~0
0
~4 It A
E I
2
I
3.2. Spectroscopy
BI
5~^
~)u
I
I
400
500
600
700
WAVELENGTH (nm)
Fig. 1. Absorption studies of the oxidation of dithionite-reduced particle flavin and cytochromes by metronidazole. The
zero absorption values show the absolute absorptions, relative
to trace A. Trace A: buffer only. Trace D: 2.0 m M metronidazole and 0.5 m M dithionite in the reference cuvette under
anaerobic conditions. Traces B, C, and E are a sequence of
spectra from one experiment with 0.88 m g / m l particle protein.
Trace B: both cuvettes anaerobic, particles reduced with 0.5
m M dithionite, and scanned after 24 rain. Trace C: 10 min
after anaerobic addition of 2.0 m M metronidazole to the
reference cuvette. Trace E: 10 min after equilibration of the
reference cuvette with oxygen. Where metronidazole (in DMSO)
was added to the reference cuvette, the same a m o u n t of D M S O
was added to the sample cuvette.
4. RESULTS
The oxidations of both ravin and cytochromes
were observed using absorption-difference spectroscopy. Electron carriers in membrane particles
of both the sample and reference cuvettes were
reduced with dithionite or N A D H . Metronidazole
addition to the reference cuvette of dithionite-reduced particles gave spectral changes expected
from oxidation of flavin and cytochrome d (Fig.
1). The absorption loss at ravin wavelengths (459
nm trough) was probably due to absorption by
oxidized ravin in the reference cuvette. Cytochrome Soret band (short wavelength) shifts can
interfere at r a v i n wavelengths and cause similar
absorption changes [10] but cytochrome interference here was minimal. This conclusion is from
the small changes at Sorer wavelengths after
oxidation with metronidazole (trace C). By contrast, oxygen addition increased Soret band absorption by about 0.2 (off scale in trace E). An
interfering chromophore develops (trace D) that
appears to be a reported minor product of
metronidazole reduction [4]. It interfered slightly
at the flavin absorption maximum and the cytochrome 553 n m - 5 6 0 nm alpha-absorption maxima
(compare traces C and E with trace D).
The apparent oxidation of cytochrome d by
metronidazole can be observed in Fig. 1, trace C.
The peak at 630 nm and trough at 650 nm are
characteristic of the reduced and oxidized bands,
respectively, of cytochrome d [11].
Absorption studies were performed with
NADH-reduced membrane particles (Fig. 2).
When metronidazole was added to the reference
137
cuvette, there were no measurable absorption
changes within 10 min (trace D). After 28 min
(trace E), absorptions corresponding to slight
oxidations of flavin and cytochrome d were observed. The difference in response to metronidazole of the dithionite-reduced particles (Fig. 1)
and the NADH-reduced particles (Fig. 2) is probably the result of the direct oxidation of the
dithionite by metronidazole [12], which eliminates
the source of reductant.
MBS caused substantial oxidation of N A D H reduced flavin (Fig. 3, trace D). There was also a
small peak at 560 nm that indicated a slight
oxidation of b-type cytochrome. A stronger oxidation of flavin and a small unassigned peak at 568
nm were the only significant differences when the
particles had been reduced with dithionite and
then oxidized by MBS (data not presented). The
MBS interfered mostly at short wavelengths (trace
A
0O-
o
/,
i,i
(~
zO
<~
oo
~r
0~30 en
<~
B
~~---------~
C
A
o-
B
2-
c
~.~
Z
m
a
O 0o~
m
&
560
-24 ~
O-
~;o
.02
~
630
/5~1
602
~;o
~;o
WAVELENGTH
650
~;o
(nm)
Figl 3 } Abso~tion studies of the o~dation of NADH-reduced
particle fla~n by MBS. Trace A: buffer only. Trace C: 5.0 mM
MBS was added to the reference cuvette. Traces B, D, and E
are a sequence of spectra with 0.91 m g / ~ particle protein in
each cuvette. Trace B: anaerobic particles 24 ~ n after
anaerobic addition of 0.1 mM N A D H to each cuvette. Trace
D: 10 ~ n after addition of 5.0 mM MBS to the reference
cuvette. Trace E: 10 ~ n after equilibration of the reference
cuvette with oxygen. Other parameters as in Fig. 1.
BA=O.O2
3_
o
('4~
6~o~
°-II
560
555-~1
5Z~
F
~
4~)0
500
E
I
650
~
I
600
WAVELENGTH
700
C) and reduction of MBS by dithionite only decreased the MBS absorption.
Oxidation of flavin by the electron acceptors
was confirmed with fluorescence spectroscopy.
Addition of either metronidazole, MBS, or oxygen
completely oxidized dithionite-reduced flavin and
the oxidations were complete within 20 s (data not
presented). Addition of either MBS or oxygen
oxidized NADH-reduced flavin but metronidazole
did not (data not presented).
(nm)
Fig. 2. Absorption studies of the oxidation of NADH-reduced
membrane particle flavin and cytochromes by metronidazole.
Trace A: buffer in the cuvettes. Trace C: 2.0 mM metronidazole added to the reference cuvette. Traces B, D, E, and F are
spectra recorded in a sequence. Trace B: both cuvettes were
loaded with anaerobic particles (0.91 m g / m l protein) and were
scanned 24 min after reduction with 0.1 mM N A D H . Traces D
and E: 10 min and 28 min, respectively, after anaerobic
addition of 2.0 mM metronidazole to the reference cuvette.
Trace F: 10 min after the reference cuvette was further oxidized
with oxygen. Other conditions as in Fig. 1.
5. DISCUSSION
Metronidazole is reduced by enzymatic reactions requiting reduced pyridine nucleotides [5,13].
N A D H - d e p e n d e n t reductive b r e a k d o w n of
metronidazole was not detectable in studies using
about two percent of the level of A. vinelandii
particle N A D H oxidase activity used here [2]. The
reaction of metronidazole with pyridine nucleo-
138
tide-reduced flavin and cytochrome was slow,
which explains the lack of reductive breakdown.
In contrast, MBS gave a strong oxidation of flavin
and an oxidation of a b-type cytochrome in
NADH-reduced particles. It has a substantial inhibitory effect on particle-catalyzed oxygen uptake [2]. These results help explain the apparent
specificity of metronidazole for low-potential
processes in this bacterium [2].
This is the first report of metronidazole causing
spectral changes characteristic of a cytochrome
oxidation. The cytochrome d has an estimated
midpoint reduction potential at pH 7.4 of +270
mV [14] and is a main terminal oxidase of the
cytochrome system [15-17]. The large difference
between the cytochrome d midpoint potential and
the midpoint potential of metronidazole ( - 4 8 6
mV at pH 7.0 [1]) indicates that metronidazole
was not oxidizing the cytochrome d. Furthermore,
there was a substantially stronger effect after dithionite reduction. Excess dithionite reduces the
metronidazole directly [12]. This indicates that a
product of metronidazole reduction is causing the
spectral changes. Some of the reductive breakdown products can act as electron acceptors [5].
However, cytochrome b is positioned prior to
cytochrome d in the electron transport sequence
[15,16] and little effect on b-type cytochrome was
observed. Alternatively the reactive species might
act as a ligand, binding to the cytochrome and
causing spectral changes. A similar spectrum is
observed with carbon monoxide binding to the
reduced cytochrome d [16,17].
These observations raise the possibility that this
pharmaceutical may have similar effects on cytochromes in other aerobic organisms. Reductive
breakdown to toxic products is decreased by
oxygen [7,12] and this is thought to partially explain the toxicity to anaerobes and resistance of
aerobes. However, high c o n c e n t r a t i o n s of
metronidazole inhibit growth of mammalian cells
[18] and aerobic microorganisms [7,19]. If reduction product formed and bound to terminal
oxidases, it might cause this inhibition. Terminal
oxidases of facultative anaerobes under microaerobic conditions might also be affected, perhaps
enhancing the effects of other cytotoxic breakdown products.
ACKNOWLEDGEMENT
This research was supported by the Achievement Foundation of Iowa State University.
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